Circular Dichroic Properties of PhosphatidylcholineLiposomes

Peter Walde* and Eveline Blochliger

Institut fur Polymere, ETH-Zentrum, Universitatstrasse 6, CH-8092 Zurich, Switzerland

Received October 21, 1996. In Final Form: December 30, 1996X

The temperature dependence of the circular dichroic properties of liposomes from different phosphati-dylcholineshas been investigatedwith theaimof clarifyingwhether circular dichroism (CD)measurementscan give information on the conformational flexibility in the region of the head group of phospholipidswithin the bilayer of liposomes. These CD measurements reflect electronic transitions of the intrinsicester chromophors localized near the chiral center of the phospholipids, and they support earlier NMRandmolecular dynamics studies which have shown that a considerable conformational rigidification in theglycerol part of the phosphatidylcholine molecules in liposomes exists. These conformational restrictionsare more significant at lower temperature, showing a significant increase in CD intensity around 220 nmon cooling the liposomes below the main phase transition temperature Tc. To probe the hydrophobicdomain of the liposomes, in which no intrinsic chromophor is localized, the achiral, linear, water insoluble1,6-diphenyl-1,3,5-hexatriene (DPH)molecule has been solubilized in liposomes. Below Tc DPH shows an(induced) CD spectrumwithmultiple maximums in the region of the absorptionmaximums of DPH (300-400 nm), the sign of the CD signal being dependent on the chirality of the phospholipids used. Above Tc,DPH in the liposomes is not CD active. On the basis of these findings, the use of the induced CD of DPHis proposed as an alternative method for the estimation of Tc of phospholipid bilayers.

Introduction

Since the initial findings by Bangham and co-workers1that closed, curved bilayers of phospholipids (calledliposomes or lipid vesicles) canbe obtained fromnaturallyoccurringphospholipids, liposomeshavebeen investigatedin many laboratories, with respect to both basic physi-cochemical properties as well as applications.2 However,although natural phospholipidsssuch as phospha-tidylcholinesare chiral molecules, with a chiral center atthe sn-2 carbon of the glycerol backbone, notmanypapersdeal with the question of how the stereoconfigurationinfluences the physicochemical properties of the ag-gregated phospholipids (bilayers, liposomes).3

In the present work we have addressed the questionwhetherchiralphosphatidylcholinemolecules in liposomesshow a circular dichroism (CD) spectrum, and if yes,whether there is a difference in the CD spectrum incomparison with nonassociated, monomeric phospha-tidylcholine molecules. From different experimentalstudies, inparticularNMR,4and frommoleculardynamicscalculations5 it is apparent that intermolecular interac-

tions within the bilayer of liposomes lead to a restrictionof all the possible conformations of phosphatidylcholine.In particular, the glycerol backbone and the relative headgroup orientation seem to be rather rigid. It is thereforethought that the chiroptical properties of phosphatidyl-choline are sensitive to such conformational restrictions.In the following, we will first report on the circular

dichroic properties of liposomes made of L-DMPC,6 L-DPPC, D-DPPC, and L-POPC.In another set of experiments, the CD spectrum of the

achiral, linear membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) solubilized within the hydrophobicbilayer region of the liposomes7 has been studied, ad-dressing the question whether an induced CD8 of DPHcan be observed due to phospholipid packing asymmetry,in analogy to what has been reported for achiral hydro-phobic dyes solubilized in synthetic chiral bilayer mem-branes.9

* To whom correspondence should be addressed.X Abstract published in Advance ACS Abstracts, February 1,

1997.(1) See for example the review: Bangham, A. D. Prog. Biophys. Mol.

Biol. 1968, 18, 31-95.(2) A selection of reviews and books on different aspects of liposomes

is: (a) Papahadjopoulos, D.; Kimelberg, H. K. Prog. Surf. Sci. 1974, 4,141-232. (b) Cullis, P. R.; Hope, M. J.; de Kruijff, B.; Verkleij, A. J.;Tilcock, C. P. S. In Phospholipids and Cellular Regulation; Kuo, J. F.,Ed.; CRC Press: Boca Raton, FL, 1985; Vol. I, pp 1-59. (c) Hauser, H.In Phospholipid Handbook; Cevc, G., Ed.; Marcel Dekker, Inc.: NewYork, 1993; pp 603-637. (d) Crommelin, D. J.; Schreier, H. InColloidalDrug Delivery Systems; Kreuter, J., Ed.; Marcel Dekker, Inc.: NewYork, 1994; pp 73-190. (e) Gregoriadis, G., Ed. Liposome Technology,2nd ed.;CRC Press: Boca Raton, FL, 1993; Vols. I-III. (f) Barenholz,Y.,Lasic,D.D.,Eds.HandbookofNonmedicalApplicationsofLiposomes;CRC Press: Boca Raton, FL, 1996; Vols. I-IV.

(3) It seems that differences in physicochemical properties (such asthe thermotropic phase transitions measured by differential scanningcalorimetry) between liposomes from enantiomerically pure phosphati-dylcholineand liposomes fromracemicmixturesaresmallbutdetectable.(a) Arnett, E. M.; Gold, J. M. J. Am. Chem. Soc. 1982, 104, 636-639.(b) Boyanov, A. I.; Tenchov, B. G.; Koynova, R. D.; Koumanov, K. S.Biochim.Biophys.Acta1983,732, 711-713. (c)Kodama,M.;Hashigami,H.; Seki, S. Biochim. Biophys. Acta 1985, 300-306. (d) Zasadzinski, J.A. N. Biochim. Biophys. Acta 1988, 946, 235-243.

(4) (a) Seelig, J.; Seelig, A. Q. Rev. Biophys. 1980, 13, 19-61. (b)Browning, J. L. In Liposomes: From Physical Structure to TherapeuticApplications; Knight, C. G., Ed.; Elsevier/North-Holland BiomedicalPress: Amsterdam, 1981; pp 189-242. (c) Strenk, L. M.; Westerman,P. W.; Doane, J. W. Biophys. J. 1985, 48, 765-773. (d) Hauser, H.;Pascher, I.; Sundell, S. Biochemsitry 1988, 27, 9166-9174. (e) Auger,M.; Van Calsteren, M.-R.; Smith, I. C. P.; Jarrell, H. C. Biochemistry1990, 29, 5815-5821. (f) Hubner, W.; Mantsch, H. H.; Paltauf, F.;Hauser, H. Biochemistry 1994, 33, 320-326. (g) Hong, M.; Schmidt-Rohr, K.; Zimmermann, H. Biochemistry 1996, 35, 8335-8341.

(5) (a) Egberts, E.; Marrink, S.-J.; Berendsen, H. J. C.Eur. Biophys.J. 1994, 22, 423-436. (b) Huang, P.; Perez, J. J.; Loew, G. H. J. Biomol.Struct., Dyn. 1994, 11, 927-956. (c) Robinson, A. J.; Richards, W. G.;Thomas, P. J.; Hann, M.M.Biophys. J. 1994, 67, 2345-2354. (d) Chiu,S.-W.; Clark, M.; Balaji, V.; Subramaniam, S.; Scott, H. L.; Jakobsson,E. Biophys. J. 1995, 69, 1230-1245.

(6) The nomenclature of phospholipids is a rather complex issue. Forthe sake of simplicity we are using here the commonly appliedabbreviationswhich donot strictly correspond to themore complicated,but recommended IUPAC-IUB shorthand designations (Silvius, J. R.In Phospholipid Handbook; Cevc, G., Ed.; Marcel Dekker, Inc.: NewYork, 1993, pp 1-22). L-DMPC, 1,2-dimyristoyl-sn-glycero-3-phospho-choline; L-DPPC,1,2-dipalmitoyl-sn-glycero-3-phosphocholine; D-DPPC,2,3-dipalmitoyl-sn-glycero-1-phosphocholine; L-POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine: D,L-DPPC, 1:1 mixture of L-DPPCand D-DPPC.

(7) (a)Andrich,M.P.;Vanderkooi, J.M.Biochemistry1976,15, 1257-1261. (b) Suurkuusk, J.; Lentz, B. R.; Barenholtz, Y.; Biltonen, R. L.;Thompson, T. E. Biochemistry 1976, 15, 1393-1401. (c) Lentz, B. R.Chem. Phys. Lipids 1989, 50, 171-190.

(8) Hatano, M. Induced Circular Dichroism in Biopolymer-DyeSystems; Spinger-Verlag: Berlin, 1986.

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S0743-7463(96)01015-3 CCC: $14.00 © 1997 American Chemical Society

Materials and Methods

L-DMPC was from Bachem, Switzerland, L-DPPC, D-DPPC,and D/L-DPPCwere fromSigma, Switzerland, L-POPCwas fromAvantiPolarLipids,USA,andDPHwas fromFluka,Switzerland.Liposomes were prepared in deionized water by the extrusion

technique,10 using polycarbonate membranes and performingfinal extrusions through filterswith 50 nmpores. For POPC theextrusion temperature was ∼25 °C, for DMPC ∼35 °C, and forDPPC∼50 °C. DPHwas solubilized in the liposome membraneby first cosolubilizing the phospholipid and DPH in the desiredratio in CHCl3. After removal of CHCl3, the liposomes wereprepared in the usual way.10 Alternatively, DPH could also besolubilized from the solid state by overlaying solid DPH withpreformedextruded liposomesundermagnetic stirring overnightat (or above) the phase transition temperature (Tc) of the lipid(see below).UV-visible absorption spectra were recorded on a Lamda 9

spectrophotometer (from Perkin-Elmer) using quartz cells witha path length of 0.1 cm. CD measurements were carried outwith a JASCO J-600 spectrophotometer using quartz cells witha path length of 0.05 cm.

Results and Discussion

The CD Spectrum of Phosphatidylcholine Lipo-somes. Small, unilamellar liposomeshavebeenpreparedfrom different phosphatidylcholines at a concentration of30 mg/mL. Figure 1a shows the room temperature CDspectrum of liposomes from L-POPC (in water) and ofmonomeric L-POPC in methanol.11Inpuremethanol, theCDsignal of L-POPC is veryweak

and difficult to measure (|[Θ]220nm| e 10 deg cm2 dmol-1),see also Colombo et al.12 In contrary, the supramolecularorganization of L-POPC within the spherical liposomesleads to a significant increase in the ellipticity around220nm(nf π* transitionsof theester chromophor,Figure1a), in analogy to phosphatidylcholine in reversemicellesandmicroemulsiongels.12 This observation indicates thatthe assembly of L-POPC into a polymolecular aggregate(irrespective whether in an organic solvent12 or in water)is paralleled by a significant increase in the CD band ofthe phospholipid. In comparison with the CD signalintensity of helical and tubular structures,13 however, themolar ellipticity is relatively small. Since L-POPC inliposomes shows aCDspectrumand L-POPC inmethanoldoes not, it is reasonable to assume the presence ofintermolecular interactions between the L-POPC mol-ecules within a liposome which restrict the number ofpossible conformations of the phospholipidmolecule. Thismaymainly be due to an asymmetric, chiral environmentwhich every L-POPC molecule experiences within theanisotropic bilayer membrane of the liposomes.On the basis of the fact that the intrinsic chromophors

in POPC are the ester groups, the conformational flex-ibility in the region of the glycerolmoiety of L-POPCseemsto be considerably reduced in liposomes in comparisonwith monomeric POPC. This is exactly what is knownfrom NMR studies: generally, it has been shown thathydrated phosphatidylcholine bilayers (small as well aslarge liposomes) adopt a preferred conformation which can be deduced from the conformation in the crystal:14 the

orientation of the head group is approximately parallel tothe bilayer surface, and the glycerol backbone is ratherrigid.4 Only a few stable conformationsswhich are inequilibrium with each othersaround the C(sn-1)-C(sn-2)bondsseem to be present.To test the generality of the circular dichroic properties

of L-POPC liposomes, further measurements have beencarried out with fully saturated phosphatidylcholines asreported in the following. The CD band of the naturally

(9) (a) Kunitake, T.; Nakashima, N.; Hayashida, S.; Yonemori, K.Chem. Lett. 1979, 1413-1416. (b) Nakashima, N.; Fukushima, H.,Kunitake, T. Chem. Lett. 1981, 1207-1210.

(10) Hope,M.J.;Bally,M.B.;Webb,G.;Cullis,P.R.Biochim.Biophys.Acta 1985, 812, 55-65.

(11) It is known from literature thatunsaturatedaswell as saturatedphosphatidylcholines do not aggregate in methanolic solution. (a)Kellaway, I.; Saunders, L.Biochim. Biophys. Acta 1970, 210, 185-186.(b) Lee, A. G.; Birdsall, N. J. M.; Levine, Y. K.; Metcalfe, J. C. Biochim.Biophys. Acta 1972, 255, 43-56.

(12) Colombo, L. M.; Nastruzzi, C.; Luisi, P. L.; Thomas, R. M.Chirality 1991, 3, 495-502.

(13) Schnur, J. M.; Ratna, B. R.; Selinger, J. V.; Singh, A.; Jyothi,G.; Easwaran, K. R. K. Science 1994, 264, 945-947.

(14) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim.Biophys. Acta 1981, 650, 21-51.

Figure 1. (a) CD spectrum of L-POPC liposomes in water (1)and of L-POPC dissolved in methanol (2). [L-POPC] ) 30 mg/mL ()39 mM), for the liposomes; and 100 mg/mL (132 mM),for the measurement in methanol. T ) 25 °C. (b) CD spectrumof liposomes from L-DPPC (1), D-DPPC (2), and a racemicmixture of L-DPPC and D-DPPC (3). [DPPC] ) 30 mg/mL ()41mM). T ) 60 °C.

Circular Dichroic Properties of Phosphatidylcholine Liposomes Langmuir, Vol. 13, No. 6, 1997 1669

occurring L-enantiomer of phosphatidylcholine in lipo-somes is always positive, while liposomes of the corre-spondingD-stereoisomers showanegative bandat around220nm, see the case of L- andD-DPPC inFigure 1b.Again,neither L- nor D-DPPC dissolved in methanol shows asignificant CD signal under the conditions used. Fur-thermore, liposomes prepared from the racemate of thephospholipidgivenoCDspectrum(Figure1b). Liposomesof L-DMPChaveagainapositiveCDbandwithamaximumaround 220 nm (not shown).Inanext set of experiments, theCDspectra of liposomes

fromL-POPC, L-DMPC, and L-DPPChave beenmeasuredas a function of temperature in order to see whether theCD spectrum is sensitive to the physical state of the alkylchains of the phospholipid within the bilayer of theliposomes. Ithasbeenknownformanyyears, inparticularfrom calorimetric studies,15,16 that below the so-called(main) phase transition temperature (Tc)swhich is acharacteristic parameter for each lipidsthe hydrocarbonchains of the two fatty acid moieties in phospholipids arein all-trans conformation and show crystalline-analogueproperties. Above Tc, in the fluid-analogue phase, transaswell asgauche conformationsarepresent. We thereforewanted toknowwhether these conformational transitionsin the hydrophobic domains of the bilayer are reflected intheCDspectrumwhich reflects the properties of the headgroup region. In otherwords,wewere interested to knowhow the physical state of the interior of the bilayerinfluences the circular dichroic properties of the headgroup (surface) area.Figure2showsthe temperaturedependenceof themolar

ellipticity at 218 nm for liposomes from L-DMPC (Tc )+24 °C),16 L-DPPC (Tc ) +41 °C),16 and L-POPC (Tc ) -3

°C).17 In the two caseswhereTc is within the temperaturerange of the experiment, a clear change in the intensityof the 220 nm CD band occurs around Tc (L-DMPC,L-DPPC). BelowTc, theCDband ismore intense ([Θ]218nm∼600-650deg cm2dmol-1) thanaboveTc ([Θ]218nm∼300-400 deg cm2 dmol-1). This observation can be understoodqualitatively on the basis of the known properties of thelipid bilayer discussed above: at a temperature higherthan Tc the phospholipids are in amore fluid state (in theso-called LR-phase, in which for example the polar headgroups occupy more space),16 allowing more and/or otherconformations around the ester chromophores than in themore crystalline lipid packing below Tc. One markeddifference can actually be seen in the overall lipidpacking: below Tc, in the so-called Lâ′-phase, the hydro-phobic chains of the phospholipid molecules are tilted atan angle of ∼30° with respect to the bilayer normal,18while in the LR-phase, the chains are perpendicular to thebilayer surface.In summary, not unexpectedly, the intrinsic CD prop-

erties of the phospholipids are therefore sensitive to thesedrastic conformational changes in the region of thehydrophobic chains. The measurement of the intrinsicCD is therefore a useful tool to detect conformationalrestrictions of phospholipids in liposomes above as wellas below Tc.Induced Circular Dichroism of DPH in Phos-

phatidylcholine Liposomes. DPH is an achiral, long,rigid, linear and hydrophobic molecule which has been(and is being) oftenused as a fluorescent anisotropy probeto monitor temperature dependent molecular order andmotionswithin liposomebilayers.7 Withinaphospholipidbilayer, at a temperature below Tc, DPH is on averageoriented parallel to the fatty acid chains and centered atroughly the ninth or tenth carbon atom away from thecarbonyl group.19 Above Tc, a more random distributionof DPH within the bilayer is obtained, explaining theobserved decrease in fluorescence polarization as thetemperature exceeds Tc.7a Literature reports that fluo-rescence polarization measurements of DPH show sharpdiscontinuities in the temperature dependence at 23 °C(for L-DMPC) and about 40-41 °C (for L-DPPC), at atemperaturewhich is almost identicalwithTc determinedcalorimetrically.7a,b

We have now addressed the question whether aninduced optical activity of DPH can be observed inliposomes due to the chirality of the phosphatidylcholinebilayer matrix within which DPH is embedded andwhether this induced CD is temperature dependent.Figure 3 shows the CD spectrum of DPH solubilized inL-DMPC liposomes at 10 and at 30 °C. While thecorresponding UV absorption spectra of DPH-containingL-DMPC liposomes (not shown) is only slightly differentat 10 °C in comparison with the spectrum recorded at 30°C, DPH shows a CD signal only in the case of the lowertemperature (10 °C, Figure 3). This induced circulardichroism is characterized by three main positive peaksat 340, 360, and 380 nm (Figure 3a). If the intensity ofthe most intense peak at 360 nm is plotted as a functionof temperature, it is seen that DPH in L-DMPC liposomesshows a CD signal only below ∼25 °C (Figure 4), belowa temperaturewhich is very close to theTc value reportedin the literature (24 °C).16 We conclude from thesemeasurements thatDPH is circular dichroic belowTc due

(15) Two recent reviews on the physical properties of liposomes: (a)Sackmann, E. InHandbook of Biological Physics, Vol I: Structure andDynamics of Membranes. From Cells to Vesicles; Lipowsky, R., Sack-mann, E., Eds.; Elsevier: Amsterdam, 1995; pp 213-304. (b) Cullis, P.R.; Fenske, D. B.; Hope, M. J. In Biochemistry of Lipids, LipoproteinsandMembranes; Vance,D.E., Vance, J.E., Eds.; Elsevier: Amsterdam,1996; pp 1-33.

(16) Lewis,R.N.A.H.;McElhaney,R.N. InTheStructureofBiologicalMembranes; Yeagle, P., Ed.; CRC Press: Boca Raton, FL, 1992; pp73-155.

(17) Lynch, D. V.; Steponkus, P. L. Biochim. Biophys. Acta 1989,984, 267-272.

(18) Blume, A. In Phospholipid Handbook; Cevc, G., Ed.; MarcelDekker, Inc.: New York, 1993; pp 455-509.

(19) (a) Heyn, M. P. FEBS Lett. 1979, 108, 359-364. (b) Jahnig, F.Proc. Natl. Acad. Sci. USA 1979, 76, 6361-6365.

Figure 2. Temperature dependence of the molar ellipticity,[Θ], at 218nmfor liposomesprepared fromL-DMPC(1), L-DPPC(2), andL-POPC(3). [L-DMPC])33mg/mL()48mM); [L-DPPC]) 35 mg/mL ()48 mM); [L-POPC] ) 31 mg/mL ()41 mM).

1670 Langmuir, Vol. 13, No. 6, 1997 Walde and Blochliger

to the decreased conformational mobility of the chiralphospholipidmoleculeswhich form the chiral, anisotropicbilayer matrix in which DPH is embedded.Similar inducedCDeffects have been observed forDPH

in the case of L-DPPC (Figure 5). Here, the criticaltemperature below which DPH shows a CD spectrum isabout 40 °C, close to the Tc of this lipid (41 °C).16 If theenantiomeric D-DPPC is used, the signal of the inducedCDofDPH is opposite, all threeCDbands around 360 nmare negative but of the same absolute intensity as in thecase of L-DPPC (Figure 5).Due to the good agreement between the temperature

dependenceof the inducedCDofDPHandthe temperaturedependence of the fluorescence depolarization measure-ments published before,7a,b it is evident that the induced

CD of DPH can be used as an alternative tool for thedetermination ofTc of liposomes from chiral phospholipidmolecules. A similar inducedCDeffect has been observedfor carotinoids incorporated into L-DMPC liposomes,20althoughnodetails on thedependency of theCDspectrumon the chirality of the phospholipid were investigated.Furthermore, in a pioneering work, Kunitake et al.9 haveused the induced CD of a hydrophobic azo dye for theestimation of Tc in chiral synthetic bilayer membranes.

Concluding Remarks

In the present work, two types of CD measurementshave been carried out. In the first set of experiments theCD signal arising from the ester chromophors of thephospholipid molecules has been measured as a functionof temperature. Since the chromophors are localized inthe region of the head group of the phospholipids, the CDspectrum reflects mainly the physicochemical propertiesof the head group area, namely, of the inner and outersurface of the liposomes. In the second series of meas-urements, an achiral, water insoluble membrane probehasbeensolubilizedand ithasbeenshownthatan inducedCD spectrum is observed only below Tc. Since DPH ismainly localized in the hydrophobic part of the bilayer,the CD properties of DPH reflect mainly the physico-chemical properties of the hydrophobic bilayer interior.Since in both types of measurements the CD spectrumshows significant changes around Tc, the chiropticalproperties of the phospholipids itself or of an addedmembrane probe can be applied as alternative method-ologies to estimate Tc of liposomes of chiral amphiphilesor to evaluate the influence of additives on Tc.

Acknowledgment. We thank Pier Luigi Luisi, SilvioBonaccio, andKenichiMorigaki for their comments. Thework has been supported by the Swiss National ScienceFoundations (CHiral 2, Grant No. 21-36734.92).

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(20) Milon, A.; Wolff, G.; Ourisson, G.; Nakatani, Y. Helv. Chim.Acta 1986, 69, 12-24.

Figure 3. CD spectrum of DPH solubilized in L-DMPCliposomes, recorded at 10 °C (1) and 40 °C (2). [L-DMPC] ) 30mg/mL ()44 mM), [DPH] ) 440 µM. The ellipticity, [Θ]λ, isreferred to the molarity of DPH.

Figure 4. Temperature dependence of the molar ellipticity,[Θ], at 360 nm of DPH in L-DMPC liposomes. [L-DMPC] ) 30mg/mL ()44 mM), [DPH] ) 440 µM. The ellipticity, [Θ]360nm,is referred to the molarity of DPH.

Figure5. InducedCDspectrumofDPH-containing liposomesof L-DPPC (1) or D-DPPC (2), recorded at 24 °C. [DPPC] ) 16mg/mL ()22 mM), [DPH] ) 220 µM. The ellipticity, [Θ]λ, isreferred to the molarity of DPH.

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